The long-range goals of this proposal are to better understand brain mechanisms underlying high alcohol- seeking behavior. The overall hypothesis to be tested is that the ventral tegmental area (VTA), nucleus accumbens (ACB), medial prefrontal cortex (MPF), basolateral amygdala (BLA) and ventral subiculum (vSUB) are involved in mediating alcohol-seeking behavior. Alcohol-seeking behavior is a result of chronic ethanol self-administration, which is expressed in the absence of alcohol, in response to cues, priming or stress. Different brain mechanisms may underlie drug-self-administration and drug-seeking behavior (reviewed in Kalivas and McFarland, 2003), although little has been done to address this difference with regard to ethanol (EtOH). Recent data from our laboratory and from other studies (Samson and Chappell 2004;Liu and Weiss 2004) support the idea that different CNS mechanisms may be regulating EtOH self- administration and EtOH-seeking behavior. The overall hypothesis will be tested using mainly the alcohol- preferring (P) line of rats;however, comparative studies will be conducted with high-alcohol-drinking (HAD) and Wistar rats. The P rat meets the criteria proposed by Cicero (1979) as a suitable animal model of alcoholism, and these rats express robust contextual cue-induced EtOH-seeking behavior (Rodd-Henricks et al, 2002a,b). Operant techniques will be used to measure EtOH self-administration and EtOH-seeking behavior. In AIM 1, c-fos and fos-B immunocytochemistry will be used to identify CNS sites and neuronal populations involved in EtOH-seeking behavior. In AIM 2, microinjection techniques in combination with operant measures of cue-induced EtOH-seeking behavior will be used to identify discrete CNS sub-regions and receptors (primarily dopamine and glutamate) involved in the expression of EtOH-seeking behavior. In AIM 3, no-net-flux and conventional microdialysis techniques will be used to assess long-term effects of chronic EtOH self-administration on dopamine (DA) neurotransmission in different VTA pathways, and the relationship between the activity of these DA systems and expression of EtOH-seeking behavior. The results of this project will provide important and novel information on CNS sub-regions, neuronal pathways, and receptors involved in EtOH-seeking behavior, which could provide new avenues for developing unique pharmacotherapeutic treatments for reducing craving and preventing alcohol relapse.